1b.Approach (from AD-416):
We will characterize novel or exotic pathogens of citrus that threaten the U.S. citrus industry. This work will include the determination of the etiology of novel diseases, characterization of the diversity present in a pathogenic taxon, the development of diagnostic tests for the pathogen, and the developent of information about disease processes and knowledge that may lead to control of the diseases. This procect will also maintain and develop an extensive collection of exotic pathogens of citrus for use by this project and collaborators from the U.S. and around the world. Detection methods will e generally PCR-based, including real-time quantitative PCR. Antibodies will also be developed. The etiology of novel diseases will be established by plant inocolulations both in the greenhouse in Beltsville and by field experiments in the countries where the disease exists. We will characterize responses of diseased plants to infections by citrus greening pathogens through the use of microarray analyses and other nucleic acid-based approaches and methodologies to identify genes whose expression is altered in infected plants compared to disease-free plants.

3.Progress Report:
We have completed the second cycle of experiments towards the objective to characterize changes in gene expression in citrus in response to infection with the citrus greening pathogen. This work is done as part of an SCA with the University of Florida. We have begun the analysis of the microarray expression data set using statistical software packages ‘R’ and ‘BioConductor’. We have also isolated RNA from citrus trees infected with Citrus chlorotic dwarf virus. Small RNA produced by the host in response to the virus has been sequenced using Illumina deep sequencing technology. Bioinformatic analysis of the sequence data has provided identification of novel virus associated with Citrus chlorotic dwarf disease. Final sequence assembly and annotation are underway. Further research on Xylella fastidiosa in sweet orange seed is ongoing, funded by a grant from USDA APHIS CPHST. We also have continued work on a project funded by the Florida citrus industry to develop antibodies against the citrus greening pathogen. We have made a ‘library’ of several million antibodies against infected insect extracts and continue the process of screening the library to identify individual antibodies with desired specificity. We have overcome a number of technical obstacles and have implemented a workable protocol to isolate an unlimited number of different antibodies against the citrus greening pathogen. We now have about 40 different antibodies in hand that bind to six different protein targets on the surface of ‘Ca. Liberibacter asiaticus’. Our results have revealed several new targets for scientific research pathways that may lead to eventual control of the disease. We have also carried out a detailed bioinformatic analysis of the genomes of the citrus greening pathogen and related bacteria.

4.Accomplishments
1.
Genome comparisons. We have completed a comprehensive comparison of the genome of ‘Ca. Liberibacter asiaticus’ with related bacteria that infect plants and animals, that are transmitted by insects, and that share the intracellular life style. This work has provided insight into adaptations of ‘Ca. Liberibacter asiaticus’ to support pathogenicity in citrus and has identified protein targets for the development of novel control methods for huanglongbing disease of citrus.